Role of an intrasubunit disulfide in the association state of the cytosolic homo-oligomer methionine adenosyltransferase.

Recombinant rat liver methionine adenosyltransferase has been refolded into fully active tetramers (MAT I) and dimers (MAT III), using as a source chaotrope-solubilized aggregates resulting from specific washes of inclusion bodies. The conditions of refolding, dialysis in the presence of 10 mm dithiothreitol or 10 mm GSH with 1 mm GSSG, allowed the production of both isoforms, the nature of the redox agent determining the capacity of the final product (MAT I/III) to interconvert. Refolding in the presence of 10 mm dithiothreitol yielded mainly MAT III in a concentration-dependent equilibrium with the homotetramer MAT I. However, refolding in the presence of the redox pair GSH/GSSG resulted in a stable MAT I and III mixture. Blockage of dimer-tetramer interconversion has been found related to the production of a single intramolecular disulfide in methionine adenosyltransferase during the GSH/GSSG folding process. The residues involved in this disulfide have been identified by mass spectrometry and using a set of single cysteine mutants as cysteines 35 and 61. In addition, a kinetic intermediate in the MAT I dissociation to MAT III has been detected. The physiological importance of these results is discussed in light of the structural and regulatory data available.